Genes with codon mutations encoding xylanase

ABSTRACT

The present disclosure provides for a polynucleotide sequences encoding a xylanase. More specifically, the present disclosure provides for polynucleotide sequences with codon mutations encoding a xylanase.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of and claims the benefit and priorityto U.S. patent application Ser. No. 14/771,181, filed on Aug. 27, 2015,which is a U.S. National Phase Application of PCT InternationalApplication Number PCT/US2014/022484, filed on Mar. 10, 2014,designating the United States of America and published in the Englishlanguage, which is an International Application of and claims thebenefit of priority to United Kingdom Patent Application No. 1308853.9,filed on May 16, 2013, and U.S. Provisional Application No. 61/777,348,filed on Mar. 12, 2013. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

FIELD OF THE INVENTION

Polynucleotide sequences encoding a xylanase are provided. Inparticular, the provided sequences provide increased expression of aspecific, thermostable, thermotolerant, pressure stable xylanase.

SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 37 C.F.R. § 1.821(e). Thename of the ASCII text file for the Sequence Listing isSeqListVEREN2004D1.txt, the date of creation of the ASCII text file isOct. 25, 2018, and the size of the ASCII text file is 5 KB.

FIELD OF THE INVENTION

This invention relates generally to enzymes, polynucleotides encodingthe enzymes, the use of such polynucleotides and polypeptides and morespecifically to enzymes having xylanase activity, e.g., endoxylanaseactivity, and/or catalyzing hydrolysis of internal β-1,4-xylosidiclinkages or endo-β-1,4-glucan linkages; and/or degrading a linearpolysaccharide beta-1,4-xylan into xylose; or, a glucanase activity,e.g., an endoglucanase activity, for example, catalyzing hydrolysis ofinternal endo-β-1,4- and/or 1,3-glucan linkages, a xylanase activity,and/or a mannanase activity.

Xylanases (e.g., endo-1,4-beta-xylanase, EC 3.218) hydrolyze internalβ-1,4-xylosidic linkages in xylan to produce smaller molecular weightxylose and xylo-oligomers. Xylans are polysaccharides formed from1,443-glycoside-linked D-xylopyranoses, Xylanases are of considerablecommercial value, being used in the food industry, for baking and fruitand vegetable processing, breakdown of agricultural waste, in themanufacture of animal feed and in pulp and paper production. Xylanasesare formed by fungi and bacteria. Arabinoxylans are major non-starchpolysaccharides of cereals representing 2.5-7.1% w/w depending onvariety and growth conditions. The physicochemical properties ofarabinoxylans polysaccharide are such that it gives rise to viscoussolutions or even gels under oxidative conditions. In addition,arabinoxylans have high water-binding capacity and may have a role inprotein foam stability. AU of these characteristics present problems forseveral industries including brewing, baking, animal nutrition and papermanufacturing. In brewing applications, the presence of xylan results inwort filterability and haze formation issues. In baking applications(especially for cookies and crackers), these arabinoxylans create stickydoughs that are difficult to machine and reduce biscuit size. Inaddition, this carbohydrate is implicated in rapid rehydration of thebaked product resulting in loss of crispiness and reduced shelf-life.For monogastric animal feed applications with cereal diets, arabinoxylanis a major contributing factor to viscosity of gut contents and therebyadversely affects the digestibility of the teed and animal growth rate.For ruminant animals, these polysaccharides represent substantialcomponents of fiber intake and more complete digestion of arabinoxylanswould facilitate higher feed conversion efficiencies.

Xylanases are used for a variety of industrial and commercial purposesincluding but not limited to: bleaching of paper pulp, increasing thebrightness of pulp, degumming plant fibers, food and feed additives,baking, extraction of coffee (Beg, Q. K.; Kapoor, M.; Mahajan, L.;Hoondal, G. S. (2001). “Microbial xylanases and their industrialapplications: A review.” Applied Microbiology and Biotechnology 56(3-4): 326-38.)

DESCRIPTION OF THE INVENTION

Enzymes are proteins that act as catalysts. Proteins are polymers ofamino acids linked in dehydration reactions by peptide bonds. Theidentity of the amino acids and the order in which they are linked toform proteins determines a given protein's activity. This order in whichamino acids are assembled into proteins (the protein “sequence”) isultimately determined by the sequence of a DNA strand which “encodes”the protein.

The three-nucleotide sequence that specifies a given amino acid to beassembled into a protein is called a “codon.” The 20 amino acids builtinto proteins are collectively encoded by 64 tri-nucleotide codonsequences. The series of codons which specifies a protein is called an“Open Reading Frame.” An amino acid may be specified by as few as one oras many as six distinct codons. A change (or mutation) in thetrinucleotide sequence of a codon that does not affect the amino acidspecified is called a “silent” mutation.

As a result, there are many DNA sequences capable of encoding the sameprotein, because the DNA sequences differ from one another only through“silent” mutations. By altering one or more of the codons which encode agiven protein, it may be possible to greatly increase the amount ofprotein which a gene produces without affecting the sequence of theprotein that is encoded.

In some embodiments, the invention comprises SEQ ID NO: 1. In someembodiments, the invention comprises the polynucleotide sequence of SEQID NO: 1. In some embodiments, this sequence encodes a protein. In someembodiments, this protein is an enzyme having xylanase activity.

The improved nucleotide sequence disclosed herein is given as SEQ ID NO:1 encodes a previously disclosed xylanase enzyme that was evolved from aparent xylanase enzyme isolated from a DNA library originating from anenvironmental sample. The disclosed xylanase is described in PCTPublication No. WO 2003/106654, SEQ ID NO: 380 (encoded by thepolynucleotide SEQ ID NO: 379 of the same publication, described hereinas SEQ ID NO: 3). In some embodiments, the invention comprises thepolynucleotide sequence of SEQ ID NO: 1, or fragments thereof. In someembodiments, these sequences encode a protein. In some embodiments, theprotein is an enzyme having xylanase activity.

The invention comprises multiple nucleotide changes with respect to SEQID NO: 3. These changes are silent as to the encoded protein. The 10base changes are set forth below. “Position” indicates the number of thenucleotide within the Open Reading Frame, with the first nucleotide ofthe first codon numbered as 1. The mutation is specified using thenotation (old nucleotide) (position) (new nucleotide). The mutations areas follows: T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C,or any combination thereof.

In some embodiments, the mutations of the present invention, weredetermined by various factors including analysis of the two dimensionaland three dimensional structure of the 5 prime end of the predicted mRNAstructure of an ORF or gene of interest (for example SEQ ID NO. 3), aswell as the preferred codons of the host, to select mutations which mayenhance the expression of the protein of interest (for example, SEQ IDNO: 4). The mutations were not selected solely on the basis of thepreferred codons of the host, that is, codon optimization, nor by acodon optimization program. As discussed in Example 1, two variants werecreated SEQ ID NO: 1 and SEQ ID NO: 2 in which silent codon mutationswere introduced based on, for example, the factors mentioned above andtested for enhanced protein expression.

The base changes which distinguish SEQ ID NO: 1 from prior reportedsequences encoding the disclosed cellulase, collectively andindividually, result in an Open Reading Frame which leads to a higherlevel of protein expression than previously employed nucleotidesequences encoding the same protein.

In some embodiments, a nucleotide sequence of a xylanase derived from anenvironmental sample is disclosed, wherein the nucleotide sequencecomprises at least one mutation selected from T6G, A12G, C15T, T33G,A42C, A48C, T57G, A66C, T69C, T72C, or any combination thereof. In someaspects of these embodiments, at least one mutation is silent as to thesequence of the encoded protein. In other aspects, at least one mutationresults in the nucleotide sequence harboring at least one mutationdirecting expression of the xylanase at a higher level than a nucleotidesequence lacking the at least one mutation and not otherwise differingfrom the nucleotide sequence of the above.

In some embodiments, a nucleotide sequence encoding a xylanase isdisclosed, wherein the nucleotide sequence comprises SEQ ID NO: 3 andhaving at least one mutation selected from T6G, A12G, C15T, T33G, A42C,A48C, T57G, A66C, T69C, T72C, or any combination thereof.

In some embodiments, a nucleotide sequence from an environmental sourceis disclosed having at least one mutation which increases the expressionlevel of a protein encoded by said nucleotide sequence compared to awild type genomic sequence. In some aspects, at least one mutation issilent.

In some embodiments, a first nucleotide sequence encoding thepolypeptide of SEQ ID NO: 4 is disclosed wherein the nucleotide sequencehas been mutated with respect to a second sequence encoding thepolypeptide of SEQ ID NO: 4 such that the expression level of theprotein is increased relative to that of the protein encoded by thesecond nucleotide sequence.

Expression Systems

In another embodiment, the DNA encoding the xylanase of the presentinvention may be introduced, either on a plasmid or stably transformedinto the genome of, for example, any number of gram negative bacterialsystems such as E. coli, Pseudomonas species such as fluorescens,Pseudomonas putida, Pseudomonas aeruginosa, Ralstonia species, orCaulobacter species. Similarly, the xylanase may be introduced into anynumber of gram positive bacterial expression systems such as Bacillusspecies such as Bacillus subtilis, Bacillus megaterium, Bacillus brevis,Lactococcus species such as Lactococcus lactis, Lactobacillus species,Streptomyces species such as Streptomyces lividans. Other gram negative,gram positive or unrelated eubacterial or archaeal expression systemsmay be used to express the xylanase.

In another embodiment, SEQ ID NO: 1 may be used to direct an increasedlevel of expression in a number of systems in which the disclosedxylanase protein may be expressed. SEQ ID NO: 1 may be introduced intoany number of expression systems to express the disclosed xylanase at animproved accumulation level. For example, SEQ ID NO: 1 may beintroduced, either on a plasmid or stably transformed into the genomeof, for example, any number of gram negative bacterial systems such asE. coli, Pseudomonas species such as fluorescens, Pseudomonas putida,Pseudomonas aeruginosa, Ralstonia species, or Caulobacter species.Similarly, SEQ ID NO: 1 may be introduced into any number of grampositive bacterial expression systems such as Bacillus species such asBacillus subtilis, Bacillus megaterium, Bacillus brevis, Lactococcusspecies such as Lactococcus lactis, Lactobacillus species, Streptomycesspecies such as Streptomyces lividans. Other gram negative, grampositive or unrelated eubacterial or archaeal expression systems may beused to express SEQ ID NO: 1. In a further embodiment, SEQ ID NO: 1 maybe introduced into any number of eukaryotic expression systems such asSaccharomyces, Schizosaccharomyces pombe, Pichia pastoris, andHansanuela polymorpha.

More specifically, SEQ ID NO: 1 may be introduced into a plasmid todirect its expression. Plasmids which SEQ ID NO: 1 may be introducedinclude, for example, E. coli expression vectors of the families pQE,pET, and pASK; Pseudomonas expression vectors of the families pCN51 LT8,RSF1010, pWZ112T, and pMYC; Bacillus expression vectors of the familiespBAX, pHT01, and pHIS1525; Streptomyces expression vectors of thefamilies 0.16021 and pIJ2460; and Lactococcus: expression vectors of thefamilies pNZ9530 and pNZ8148, for example. These examples are fordemonstrative purposes and do not represent a complete set of vectors inwhich the polynucleotide sequence of SEQ ID NO: 1 can be expressed.

In another embodiment, the expression system can be any Pseudomonasfluorescens expression system known in the art, for example, thePseudomonas fluorescens expression system that is commercially availablefrom Dow Global Technologies Inc., strain DC454 (US Patent PUB. APP. NO.20050130160 and US Patent PUB. APP. NO. 20050186666). A nucleic acidsequence encoding the xylanase enzyme or polypeptide is inserted eitherin the pMYC vector (Dow Global Technologies Inc., US Patent PUB. APP.NO. 20050130160) or in the pDOW1169 vector (Dow Global TechnologiesInc., US Patent PUB. APP. NO. 20080058262) and then introduced into thePseudomonas fluorescens host by electroporation. Those skilled in theart will know alternative vectors that can be used as embodiments ofthis invention.

In some embodiments, the xylanase will be expressed at least at thefollowing expression levels: 65 U/ml, 70 U/ml, 75 U/ml, 80 U/ml, 85U/ml, 90 U/ml, 95 U/ml, 100 U/ml, 105 U/ml, 110 U/ml, 115 U/ml, 120U/ml, 125 U/ml, 130 U/ml, 135 U/ml, 140 U/ml, 145 U/ml, 150 U/ml, 155U/ml, 160 U/ml, 165 U/ml, 170 U/ml, 175 U/ml, 180 U/ml, or more.

Nucleic Acids

The invention also provides isolated, synthetic, or recombinant nucleicacids comprising sequences completely complementary to the nucleic acidsequences of the invention (complementary (non-coding) and codingsequences also hereinafter collectively referred to as nucleic acidsequences of the invention).

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid encoding at least one polypeptide having axylanase activity, wherein the nucleic acid comprises a sequence havingat least about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%. 85%, 86%, 87%, 88%,89%, 90%, 91%, 97%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, orcomplete (100%) sequence identity (homology) to an exemplary nucleic addof the invention, including the sequence of SEQ ID NO: 1. For example,the invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid sequence SEQ ID NO: 1 (the exemplarypolynucleotide sequences of this invention). The invention providesisolated, synthetic, or recombinant nucleic acids encoding a polypeptidecomprising a sequences as set forth in SEQ ID NO: 4 (the exemplarypolypeptide sequences of this invention), and enzymatically activefragments thereof.

Polypeptide

Polypeptides and peptides of the invention are isolated, synthetic, orrecombinant polypeptides. Peptides and proteins can be recombinantlyexpressed in vitro or in vivo. The peptides and polypeptides of theinvention can be made and isolated using any method known in the art.Polypeptides and peptides of the invention can also be synthesized,whole or in part, using chemical methods well known in the art. Forexample, xylanase polypeptides can be produced in a standard recombinantexpression system (as described herein), chemically synthesized, orpurified from organisms in which they are naturally expressed.

The invention provides isolated, synthetic or recombinant polypeptideshaving a xylanase activity, comprising an amino acid sequence having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,67%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 97%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100%(complete) sequence identity to an exemplary amino acid sequence of theinvention (e.g., SEQ ID NO: 4), or an enzymatically active fragmentthereof.

The invention provides isolated, synthetic or recombinant polypeptidescomprising a sequence as set forth in SEQ ID NO: 4, and enzymaticallyactive fragments thereof, and variants thereof.

In some embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having a xylanase activity but lacking asignal sequence, a prepro domain, a dockerin domain, and/or acarbohydrate binding module (CBM); and in one aspect, the carbohydratebinding module (CBM) comprises, or consists of a xylan binding module, acellulose binding module, a lignin binding module, a xylose bindingmodule, a mannose binding module, a xyloglucan-specific module and/or aarabinofuranoside binding module.

In some embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having a xylanase activity furthercomprising a heterologous sequence; and in one aspect, the heterologoussequence comprises, or consists of a sequence encoding: (i) aheterologous signal sequence, a heterologous carbohydrate bindingmodule, a heterologous dockerin domain, a heterologous catalytic domain(CD), or a combination thereof; (ii) the sequence of (i), wherein theheterologous signal sequence, carbohydrate binding module or catalyticdomain (CD) is derived from a heterologous enzyme; or, (iii) a tag, anepitope, a targeting peptide, a cleavable sequence, a detectable moietyor an enzyme; and in one aspect, the heterologous carbohydrate bindingmodule (CBM) comprises, or consists of, a xylan binding module, acellulose binding module, a lignin binding module, a xylose bindingmodule, a mannose-binding module, a xyloglucan-specific module and/or aarabinofuranoside binding module; and in one aspect, the heterologoussignal sequence targets the encoded protein to a vacuole, theendoplasmic reticulum, a chloroplast or a starch granule.

Thermostability

In one aspect, the recombinant nucleic acid of the present inventionencodes a polypeptide having a xylanase activity that is thermostable.For example, a polypeptide of the invention, SEQ ID NO 4, or the variantevolved enzymes of the invention can be thermostable. The thermostablepolypeptide according to the invention can retain binding and/orenzymatic activity e.g., xylanase activity, under conditions comprisinga temperature range from greater than 37° C. to about 95° C., or betweenabout 55° C. to about 85° C., or between about 70° C. to about 75° C.,or between about 70° C. to about 95° C., between about 90° C. to about95° C., between about 95° C. to about 105° C., or between about 95° C.to about 110° C. in one aspect, wherein the polypeptide can retainbinding and/or enzymatic activity, e.g., xylanase activity, underconditions comprising 1° C. to about 5° C., between about 5° C. to about15° C., between about 15° C.: to about 25° C., between about 25° C. toabout 37° C. In one aspect polypeptides of the invention can retainbinding and/or enzymatic activity, e.g., xylanase activity, underconditions comprising 90° C., 91° C., 92° C., 93° C., 94° C., 95° C.,96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103°C., 104° C., 105° C., 107° C., 108° C., 109° C., or 110° C., or more. Insome embodiments, the thermostable polypeptides according to theinvention retains activity, e.g., a xylanase activity at a temperaturein the ranges described above, under acidic conditions comprising aboutpH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, or pH 4 or less (more acidic), or,retain a xylanase activity under acidic conditions comprising about pH6.5, pH 6, pH 5.5, pH 5, pH 4.5, or pH 4 or less (more acidic); or,retain xylanase activity under basic conditions comprising about pH 7,pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH12, pH 12.5 or more (more basic) or, retain a xylanase activity underbasic conditions comprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (morebasic).

Thermotolerant

In one aspect, the recombinant nucleic acid of the present inventionencodes a polypeptide having a xylanase activity that is thermotolerant.For example, a polypeptide of the invention, SEQ ID NO: 4, or thevariant evolved enzymes of the invention can be thermotolerant. In oneaspect, the xylanase activity is thermotolerant, e.g., wherein thepolypeptide retains xylanase activity alter exposure to a temperature inthe range from greater than 37° C. to about 95° C., or between about 55°C. to about 85° C., or between about 70° C. to about 75° C., or betweenabout 70° C. to about 95° C., between about 90° C. to about 9555° C.,between about 95° C. to about 105° C., or between about 95° C. to about110° C. In one aspect, wherein the polypeptide retains a xylanaseactivity after exposure to conditions comprising a temperature range ofbetween about 1° C. to about 55° C., between about 5° C. to about 1.5°C., between about 15° C. to about 25° C., between about 25° C. to about37° C. In one aspect polypeptides of the invention can retain a xylanaseactivity after exposure to a temperature up to 90° C., 91° C., 92° C.93° C. 94° C., 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C.,102° C. 103° C., 104° C. 105° C., 107° C., 108° C., 109° C., or 110° C.,or more. In one aspect, the xylanase activity of polypeptides encoded bynucleic acids of the invention retain activity after exposure to acidicconditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, or pH 4or less (more acidic), or, retain a xylanase activity after exposure toacidic conditions comprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5,or pH 4 or less (more acidic); or, retain activity under basicconditions comprising about pH 7, pH 7.5, pH 8.0, pH 8.5, pH 9, pH 9.5,pH 10, pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic) or,retain a xylanase activity after exposure to basic conditions comprisingabout pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11,pH 11.5, pH 12, pH 12.5 or more (more basic).

Cellulosic Digestion

Xylanases of the invention are particularly useful in baking, animalfeed, beverage and wood, wood pulp, Kraft pulp, paper, paper product orpaper pulp processes. In one aspect, an enzyme of the invention isthermotolerant and/or tolerant of high and/or low pH conditions. Forexample, in one aspect, a xylanase, a mannanase and/or a glucanase ofthe invention retains activity under conditions comprising a temperatureof at least about 80° C., 85° C., 86° C., 87° C., 88° C., 89° C., 90°C., 91° C., 92° C., 93° C., 94° C., 95° C., 90° C., 97° C., 98° C., 99°C., 100° C., 101° C., 102° C., 103° C., 103.5° C., 104° C., 105° C.,107° C. 108° C., 109° C. or 110° C., or more, and a basic pH of at leastabout pH 11, or more.

The invention provides industrial, agricultural or medical applications:e.g., biomass to biofuel, e.g., ethanol, propanol, butanol and/ormethanol, using enzymes of the invention having decreased enzyme costs,e.g., decreased costs in biomass to biofuel conversion processes. Thus,the invention provides efficient processes for producing bioalcohols,biofuels and/or biofuel- (e.g., bioethanol-, propanol-, butanol- and/ormethanol-) comprising compositions, including synthetic, liquid or gasfuels comprising a bioalcohol, from any biomass.

In one aspect, enzymes of the invention, including the enzyme“cocktails” of the invention (“cocktails” meaning mixtures of enzymescomprising at least one enzyme of this invention), are used to hydrolyzethe major components of a lignocellulosic biomass, or any compositioncomprising cellulose and/or hemicellulose (lignocellulosic biomass alsocomprises lignin), e.g., seeds, grains, tubers, plant waste (such as ahay or straw, e.g., a rice straw or a wheat straw, or any the dry stalkof any cereal plant) or byproducts of food processing or industrialprocessing (e.g., stalks), corn (including cobs, stover, and the like),grasses (e.g., Indian grass, such as Sorghastrum nutans; or, switchgrass, e.g., Panicum species, such as Panicum virgatum), wood (includingwood chips, processing waste, such as wood waste), paper, pulp, recycledpaper (e.g., newspaper); also including a monocot or a dicot, or amonocot corn, sugarcane or parts thereof (e.g., cane tops), rice, wheat,barley, switchgrass or Miscanthus; or a dicot oilseed crop, soy, canola,rapeseed, flax, cotton, palm oil, sugar beet, peanut, tree, poplar orlupine; or, woods or wood processing byproducts, such as wood waste,e.g., in the wood processing, pulp and/or paper industry, in textilemanufacture and in household and industrial cleaning agents, and/or inbiomass waste processing.

Dietary

In one embodiment, the xylanase of the present invention may be used topre-treat, modify, or digest a food, food additive, or dietarysupplement for animals or human beings. In one embodiment, the xylanaseof the present invention may be used as a food, food additive, ordietary supplement for animals or human beings. In one aspect thexylanase will treat or will act as a prophylaxis for digestivedisorders. In another aspect of the present invention the xylanase willalter or enhance digestion. In another aspect of the present inventionthe xylanase will enhance, alter, or aid in the digestion of foodstuffs.In a further aspect of the invention the xylanase will enhance, aid, oralter in the nutrient value of foodstuffs. In a further aspect, thexylanase is active in the digestive tract, e.g., in a stomach and/orintestine.

In another embodiment, the xylanase of the invention may be used as ananimal feed or an animal feed additive. In another embodiment thethermostability and/or thermotolerance of the xylanase allows for theformation of pellets without the need for a secondary agent such as saltor wax. An animal feed comprising a xylanase can be provided to ananimal in any formulation known to those skilled in the art. Examples ofanimal feed formulations include, but are not limited to: a deliverymatrix, a pellet, a tablet, a gel, a liquid, a spray, ground grain, or apowder.

The invention provides edible enzyme delivery matrix comprising athermostable recombinant xylanase, e.g., a polypeptide of the invention.The invention provides methods for delivering a xylanase to an animal,the method comprising: preparing an edible enzyme delivery matrix in theform of pellets comprising a granulate edible carrier and a thermostablerecombinant xylanase, a mannanase and/or a glucanase enzyme, wherein thepellets readily disperse the xylanase, a mannanase, and/or a glucanaseenzyme contained therein into aqueous media, and administering theedible enzyme delivery matrix to the animal. The recombinant xylanaseenzyme can comprise a polypeptide of the invention. The granulate ediblecarrier can comprise a carrier selected from the group consisting of agrain germ, a grain germ that is spent of oil, a hay, an alfalfa, atimothy, a soy hull, a sunflower seed meal and a wheat midd. The ediblecarrier can comprise grain germ that is spent of oil. The xylanaseenzyme can be glycosylated to provide thermostability at pelletizingconditions. The delivery matrix can be formed by pelletizing a mixturecomprising a grain germ and a xylanase. The pelletizing conditions caninclude application of steam. The pelletizing conditions can compriseapplication of a temperature in excess of about 80° C. for about 5minutes and the enzyme retains a specific activity of at least 350 toabout 900 units per milligram of enzyme.

Methods of Making Ethanol

The invention provides methods for making ethanol comprising contactinga starch—comprising composition with a polypeptide having a xylanaseactivity, wherein the polypeptide has a sequence of the invention, orthe polypeptide is encoded by a nucleic acid comprising a sequence ofthe invention, or an enzymatically active fragment thereof. Theinvention provides compositions comprising a starch and a polypeptidehaving a xylanase activity, wherein the polypeptide has a sequence ofthe invention, or the polypeptide is encoded by a nucleic acidcomprising a sequence of the invention, or an enzymatically activefragment thereof.

Brewing and Fermenting

The invention provides methods of brewing (e.g., fermenting) beercomprising the xylanase of the invention. In one exemplary process,starch-containing raw materials are disintegrated and processed to forma malt. An enzyme of the invention is used at any point in thefermentation process. The xylanase of the invention can be used in thebrewing industry for the degradation of beta-glucans. In one aspect, thexylanase of the invention are used in the brewing industry for theclarification of the beverage. Enzymes of the invention can be used inthe beverage industry in improving filterability of wort or beer, asdescribed, e.g., in U.S. Pat. No. 4,746,517.

In one aspect, the xylanase of the invention can be used in theprocessing of barley malt. The major raw material of beer brewing isbarley malt. This can be a three stage process. First, the barley graincan be steeped to increase water content, e.g., to around about 40%.Second, the grain can be germinated by incubation at 15 to 25° C. for 3to 6 days when enzyme synthesis is stimulated under the control ofgibberellins. In one aspect, enzymes of the invention are added at this(or any other) stage of the process.

In one aspect, enzymes of the invention are used in mashing andconversion processes. In the brewing and fermentation industries,mashing and conversion processes are performed at temperatures that aretoo low to promote adequate degradation of water-soluble glucans,mannans, arabinoxylans or xylans, or other polysaccharides. Thesepolymers form gummy substrates that can cause increased viscosity in themashing wort, resulting in longer mash run-off, residual haze andprecipitates in the final beer product due to inefficient filtration andlow extraction yield.

In one aspect, enzymes of the invention are used in malthouseoperations, e.g., glucanase is added to the process water, to shortengermination times and/or to encourage conversion of poor quality barleyto acceptable malts. In one aspect, enzymes of the invention are usedfor mashing, e.g., they are added to increase wort filterability and/orimprove lautering (separating the wort from the mash). In one aspect,enzymes of the invention are used in the fermentor and/or settling tankto, e.g., assist in haze clearing and/or to improve filtration. In oneaspect, enzymes of the invention are used in adjunct brewing, e.g., aglucanase of the invention is added to breakdown glucans, mannans,arabinoxylans or xylans, or other polysaccharides from barley, wheat,and/or other cereals, including glycans in malt. In one aspect, enzymesof the invention are used in malt brewing, e.g., a glucanase of theinvention is added to modify poor malts with high glucan content.

Glucanases, (or cellulases), mannanases, xylanases, amylases,xanthanases and/or glycosidases, e.g., cellobiohydrolases, mannanasesand/or beta-glucosidases of the invention can be used in any beer oralcoholic beverage producing process, as described, e.g., in U.S. Pat.Nos. 5,762,991; 5,536,650; 5,405,624; 5,021,246; 4,788,066.

Treating Foods and Food Processing

The xylanase of the invention has numerous applications in foodprocessing industry. For example, in one aspect, the enzymes of theinvention are used to improve the extraction of oil from oil-rich plantmaterial, e.g., oil-rich seeds, for example, soybean oil from soybeans,olive oil from olives, rapeseed oil from rapeseed and/or sunflower oilfrom sunflower seeds.

The xylanase of the invention can be used for separation of componentsof plant cell materials. For example, enzymes of the invention can beused in the separation of glucan-rich material (e.g., plant cells) intocomponents. In one aspect, enzymes of the invention can be used toseparate glucan-rich or oil-rich crops into valuable protein and oil andhull fractions. The separation process may be performed by use ofmethods known in the art.

The xylanase of the invention can be used in the preparation of fruit orvegetable juices, syrups, extracts and the like to increase yield. Theenzymes of the invention can be used in the enzymatic treatment (e.g.,hydrolysis of glucan-comprising plant materials) of various plant cellwall-derived materials or waste materials, e.g. from cereals, grains,wine or juice production, or agricultural residues such as vegetablehulls, bean hulls, sugar beet pulp, olive pulp, potato pulp, and thelike. The enzymes of the invention can be used to modify the consistencyand appearance of processed fruit or vegetables. The enzymes of theinvention can be used to treat plant material to facilitate processingof plant material, including foods, facilitate purification orextraction of plant components. The xylanase of the invention can beused to improve feed value, decrease the water binding capacity, improvethe degradability in waste water plants and/or improve the conversion ofplant material to ensilage, and the like. The xylanase of the inventioncan also be used in the fruit and brewing industry for equipmentcleaning and maintenance.

Detergent Compositions

The invention provides detergent compositions comprising one or morepolypeptides of the invention and methods of making and using thesecompositions. The invention incorporates all methods of making and usingdetergent compositions, see, e.g., U.S. Pat. Nos. 6,413,928; 6,399,561;6,365,561; 6,380,147, The detergent compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The invention also provides methodscapable of a rapid removal of gross food soils, films of food residueand other minor food compositions using these detergent compositions.Enzymes of the invention can facilitate the removal of starchy stains bymeans of catalytic hydrolysis of the starch polysaccharide. Enzymes ofthe invention can be used in dishwashing detergents in textilelaundering detergents. The actual active enzyme content depends upon themethod of manufacture of a detergent composition and is not critical,assuming the detergent solution has the desired enzymatic activity. Inone aspect, the amount of glucosidase present in the final solutionranges from about 0.001 mg to 0.5 mg per gram of the detergentcomposition. The particular enzyme chosen for use in the process andproducts of this invention depends upon the conditions of final utility,including the physical product form, use pH, use temperature, and soiltypes to be degraded or altered. The enzyme can be chosen to provideoptimum activity and stability for any given set of utility conditions.In one aspect, the polypeptides of the present invention are active inthe pH ranges of from about 4 to about 12 and in the temperature rangeof from about 20° C. to about 95° C. The detergents of the invention cancomprise cationic, semi-polar nonionic, or zwitterionic surfactants; or,mixtures thereof.

The present invention provides cleaning compositions including detergentcompositions for cleaning hard surfaces, detergent compositions forcleaning fabrics, dishwashing compositions, oral cleaning compositions,denture cleaning compositions, and contact lens cleaning solutions. Inone aspect, the invention provides a method for washing an objectcomprising contacting the object with a polypeptide of the inventionunder conditions sufficient for washing. A polypeptide of the inventionmay be included as a detergent additive. The detergent composition ofthe invention may, for example, be formulated as a hand or machinelaundry detergent composition comprising a polypeptide of the invention.A laundry additive suitable for pre-treatment of stained fabrics cancomprise a polypeptide of the invention. A fabric softener compositioncan comprise a polypeptide of the invention. Alternatively, apolypeptide of the invention can be formulated as a detergentcomposition for use in general household hard surface cleaningoperations.

DESCRIPTION OF THE FIGURES

FIG. 1 is an image of SDS PAGE gel electrophoresis displaying variouslevels of protein expression, as described in Example 2.

FIG. 2 is a bar graph showing the level of activity of proteinpreparations, as described in Example 3.

FIG. 3 is SEQ ID NO: 1, the polynucleotide with 10 silent mutations:T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, as comparedto SEQ ID NO: 3.

FIG. 4 is SEQ ID NO: 2, the polynucleotide with 10 silent mutations: ascompared to SEQ ID NO: 3.

FIG. 5 is SEQ ID NO: 3, the unmodified parent polynucleotide sequence ofSEQ ID NO: 1 and 2.

FIG. 6 is SEQ ID NO: 4, the polypeptide encoded by SEQ ID NO: 1, 2, and3.

FIG. 7 is a sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 3.

FIG. 8 is a sequence alignment of SEQ ID NO: 2 and SEQ ID NO: 3.

TERMS TO DEFINE

A “codon” is a three polynucleotide sequence that specifies the identityof an amino acid to be added to a protein.

A “silent mutation” is a mutation in a codon that does not result in thespecification of a different amino acid.

An “Open Reading Frame” is a series of codons that specifies thesequence of amino acids in a protein.

A base “position” is the numerical location of a base in apolynucleotide sequence, counted consecutively from the start of theopen reading frame or from some other reference marker.

To “encode” a protein means to specify the amino acid sequence of thatprotein.

“Codon optimization” as used herein refers to modifying the codons of agene or ORF to those that are more frequently or most frequently used inthe target and host organisms, but does not alter the amino acidsequence of the original translated protein. An examples of codonoptimization software is Aptagen's Gene Forge® codon optimization andcustom gene synthesis platform (Aptagen, Inc., 2190 Fox Mill Rd. Suite300, Herndon, Va. 20171). Other publicly available databases foroptimized codons are available and can work equally in some embodimentsas well.

A “mutation” is a change in a nucleotide sequence or an amino acidsequence compared to a reference.

A “nucleotide” refers to one of the four bases which comprise DNAsequence—Adenine (A), Thymidine (T), Guanidine (G), and Cytosine (C).

“Xylanase” is an enzyme with xylanase, mannanase, and/or a glucanaseactivity.

A “Unit” (abbreviated as U) is defined as 1 μmole of reducing sugars perminute at pH 6.5 and 40° C.

Example 1

Method of making enhanced expression variants.

Two variants (SEQ ID NO: 1 and NO: 2) were designed based on SEQ ID NO:3 to mutate at the DNA level to improve the gene expression. The designof the two mutant variants takes into account the two dimensional, threedimensional structures of the DNA sequences, as well as the preferredcodons of the host, all of which that may influence gene expression. Themutations were introduced on the PCR primers. Both genes werePCR-amplified and clones into the Pseudomonas vector pDOW1169 (DOWAgroSciences, IN) using standard molecular cloning techniques. Theresulting expression constructs were transformed into Pseudomonasfluorescens DC454 (DOW AgroSciences, IN) and expression levels of theproteins were determined. A transformant with the SEQ ID NO:1 wasdesignated as the lead showing significantly enhanced expression levelsover the parent SEQ ID NO: 3.

Example 2

Method of preparing SDS-PAGE to visually determine expression levels ofvariants. Criterion™ precast Tris-HCl polyacrylamide gel (Bio-radLaboratories, Inc) was used to separate proteins. The gel was run at150V using Tris-glycine buffer (FIG. 1). Protein loading was normalizedto load proteins from 0.33 OD600 cells for each lane. SeeBlue®pre-stained protein standard was used (Life Technologies). As shown inFIG. 1, the expression level of SEQ ID NO: 2 was lower than theexpression level of parent SEQ ID NO: 3, and SEQ ID NO: 1.

Example 3

Method of determining relative expression levels for variants.

SEQ ID NO: 1, 2, and 3 genes were expressed in shake flask. The cultureswere grown at 30° C. and 220 rpm to an OD600 of ˜19 in a designedcomplex medium, and induced with 0.3 mM IPTG(Isopropylβ-D-1-thiogalactopyranoside) for 24 hours. Cells wereharvested and lysed either by sonication or heat-treatment at 65° C. for1 hour. Xylanase activity was measured by Nelson-Somogyi reducing sugarassay using wheat arabinoxylan as substrate. (Roger A. O'Neill, AlanDarvill, Peter Albersheim, A fluorescence assay for enzymes that cleaveglycosidic linkages to produce reducing sugars, Analytical Biochemistry,Volume 177, Issue 1, (1989)11-15). Activity levels were measured in U/mlas shown in FIG. 2 to determine relative expression levels from eachculture. A Unit (abbreviated as U) is defined as 1 μmole of reducingsugars per minute at pH 6.5 and 40° C. The specific activity for theassay in this assay was determined to be 533 units/mg protein. As shownin FIG. 2, the expression level of SEQ ID NO: 2 was lower than theexpression level of parent SEQ ID NO: 3, and SEQ ID NO: 1.

What is claimed is:
 1. An animal feed comprising a polypeptide encodedby a polynucleotide comprising a nucleic acid with the sequence of SEQID NO: 1, wherein said polypeptide has xylanase activity.
 2. An animalfeed comprising a polypeptide encoded by a polynucleotide comprising anucleic acid sequence having the sequence of SEQ ID NO: 3 with theexception of at least one mutation selected from the group consisting ofT6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C, T72C, and anycombination thereof, wherein said polypeptide has xylanase activity. 3.An animal feed comprising a polypeptide encoded by a polynucleotidecomprising a nucleic acid sequence, wherein said nucleic acid sequencecomprises a sequence exhibiting at least 90%, 95%, 97%, 98%, 99%, or100% sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2, and wherein saidnucleic acid sequence comprises at least one mutation selected from thegroup consisting of T6G, A12G, C15T, T33G, A42C, A48C, T57G, A66C, T69C,T72C, and any combination thereof, relative to SEQ ID NO:1 or SEQ ID NO:2, respectively.
 4. The animal feed of claim 1, 2 or 3, wherein thepolypeptide having xylanase activity is produced in a recombinantbacterial expression system.
 5. The animal feed of claim 4, wherein thebacterial expression system is (a) a gram-negative bacteria expressionsystem, (b) the gram-negative bacteria expression system of (a), whereinthe gram-negative bacteria expression system is a Pseudomonas, E. coli,Ralstonia, or Caulobacter expression system, or (c) the gram-negativebacteria expression system of (b), wherein the Pseudomonas expressionsystem is a Pseudomonas fluorescens expression system.
 6. The animalfeed of claim 5, wherein the xylanase that is produced is at least: 65U/ml, 70 U/ml, 75 U/ml, 80 U/ml, 85 U/ml, 90 U/ml, 95 U/ml, 100 U/ml,105 U/ml, 110 U/ml, 115 U/ml, 120 U/ml, 125 U/ml, 130 U/ml, 135 U/ml,140 U/ml, 145 U/ml, 150 U/ml, 155 U/ml, 160 U/ml, 165 U/ml, 170 U/ml,175 U/ml, or 180 U/ml.
 7. The animal feed of claim 4, wherein thepolypeptide: (a) does not have a signal sequence, a proprotein sequence,or any combination thereof, or (b) further comprises a heterologousamino acid sequence selected from the group consisting of: a signalsequence, a tag, an epitope, an N-terminal extension, a C-terminalextension, and any combination thereof.
 8. The animal feed of claim 4,wherein: (a) the polypeptide further comprises at least a second enzyme,or (b) the polypeptide of (a), wherein the second enzyme is selectedfrom the group consisting of: a lactase, a lipase, a protease, acatalase, a xylanase, a cellulase, a glucanase, a mannanase, an amylase,an amidase, an epoxide hydrolase, an esterase, phospholipase,transaminase, an amine oxidase, cellobiohydrolase, an ammonia lyase, andany combination thereof.
 9. The animal feed of claim 2, wherein thepolynucleotide has the mutation T6G.
 10. The animal feed of claim 2,wherein the polynucleotide has the mutation A12G.
 11. The animal feed ofclaim 2, wherein the polynucleotide has the mutation C15T.
 12. Theanimal feed of claim 2, wherein the polynucleotide has the mutationT33G.
 13. The animal feed of claim 2, wherein the polynucleotide has themutation A42C.
 14. The animal feed of claim 2, wherein thepolynucleotide has the mutation A48C.
 15. The animal feed of claim 2,wherein the polynucleotide has the mutation T57G.
 16. The animal feed ofclaim 2, wherein the polynucleotide has the mutation A66C.
 17. Theanimal feed of claim 2, wherein the polynucleotide has the mutationT69C.
 18. The animal feed of claim 2, wherein the polynucleotide has themutation T72C.
 19. A method of making an animal feed, the methodcomprising: (a) contacting a xylanase with an animal feed, wherein thexylanase is: (i) a xylanase encoded by a polynucleotide comprising anucleic acid with the sequence of SEQ ID NO: 1; (ii) a xylanase encodedby a polynucleotide comprising a nucleic acid sequence having thesequence of SEQ ID NO: 3 with the exception of at least one mutationselected from the group consisting of T6G, A12G, C15T, T33G, A42C, A48C,T57G, A66C, T69C, T72C, and any combination thereof; or (iii) a xylanaseencoded by a polynucleotide comprising a nucleic acid sequence, whereinsaid nucleic acid sequence comprises a sequence exhibiting at least 90%,95%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1 or SEQ IDNO: 2, and wherein said nucleic acid sequence comprises at least onemutation selected from the group consisting of T6G, A12G, C15T, T33G,A42C, A48C, T57G, A66C, T69C, T72C, and any combination thereof,relative to SEQ ID NO:1 or SEQ ID NO: 2, respectively.
 20. The method ofclaim 19, wherein the animal feed is in the form of a pellet.
 21. Amethod of feeding an animal, the method comprising providing the animalfeed made by the method of claim 19 to an animal.